Epigenetic modifications such as DNA methylation play a key role in regulating gene expression. Emerging evidence suggests that intermediates generated during DNA demethylation may have distinct biological roles. However, their detection remains challenging due to their low abundance. Now, researchers from Japan have developed a novel light-sensitive oligonucleotide probe that selectively crosslinks with 5-formylcytosine, an epigenetically important intermediate, enabling its detection in target DNA and complex biological samples.
Epigenetic modifications, which are reversible changes to DNA, control when and where genes are switched on or off, playing a critical role in human growth and disease development. DNA methylation is one such key mechanism that adds methyl groups to DNA. These methyl groups are more commonly added at the 5-position of cytosine to form 5-methylcytosine (5mC), localized at gene promoter regions, resulting in gene silencing. Conversely, demethylation or the removal of methyl groups activates gene transcription.
Mounting evidence suggests that the cytosine intermediates generated during the oxidation and subsequent demethylation of 5-position of cytosine may serve distinct epigenetic roles in biological processes. Detecting and understanding these intermediates may open new avenues for disease management. However, the low abundance of these intermediates makes their detection difficult.
To overcome this challenge, a research team led by Professor Asako Yamasoshi from the Department of Life Science and Technology, Institute of Science Tokyo (Science Tokyo), Japan, has developed a novel photochemical sensor that can detect cytosine derivatives using light. Their findings were made available online on January 07, 2026, and published in Volume 148, Issue 2 of the Journal of the American Chemical Society on January 21, 2026. The researchers demonstrate the selective crosslinking of a light-sensitive oligonucleotide probe with 5-formylcytosine (5fC), a 5mC derivative central to demethylation and epigenetic regulation.
"Our work introduces a new concept for light-driven detection of 5fC, offering a spatiotemporally controllable probe for epigenetic analysis," explains Yamasoshi.
The team designed and synthesized oligonucleotide probes containing trioxsalen—a psoralen (Ps) derivative—which is a natural compound that can insert itself into DNA. The Ps-conjugated oligonucleotides undergo "photo-cycloaddition" or crosslink with the target DNA upon exposure to ultraviolet (UV) radiation at 365 nm. The researchers previously used these Ps-oligos to detect oncogenic mutations and epigenetic modifications.
In the current study, they assessed the photo-crosslinking efficiency of the probes tagged with a fluorophore, combined with different cytosine derivatives in the target DNA. Notably, the fluorescence intensity was the highest for 5fC compared to the other derivatives such as 5-hydroxymethylcytosine (5hmC) and 5-carboxylcytosine (5caC). Further, the cross-linking efficiency between the probe and 5hmC or 5caC decreased significantly as sodium-ion concentration and temperature were reduced. Conversely, cross-linking between the probe and 5fC remained largely unchanged across ionic and temperature variations, indicating a more stable interaction.
In contrast to photo-cycloaddition, exposure to shorter-wavelength UV radiation can induce "cycloreversion" by weakening interactions between the target DNA and the probe. To assess stability, the researchers irradiated the cross-linked products with UV radiation at 254 nm. Notably, the fluorescence intensity of the probe and 5fC crosslinked product remained unchanged, whereas a decrease was observed for the other cytosine derivatives, indicating greater stability of 5fC.
Finally, the researchers demonstrated the practical feasibility of 5fC detection by constructing a DNA chip sensor fabricated with the oligonucleotide probe. They observed strong fluorescence for 5mC and 5fC after crosslinking. Additionally, the fluorescence intensity of target 5mC reduced substantially after applying UV radiation at 254 nm, whereas that of 5fC remained constant, highlighting selective photo-reactivity of the probe towards 5fC .
Overall, these findings highlight the unique potential of the photo-crosslinkable oligonucleotide probe in the selective detection of 5fC in the target DNA. "We aim to extend the method to complex biological samples and improve detection sensitivity by enriching 5fC-containing DNA fragments, ultimately translating the technology into research and diagnostic tools across life sciences and medicine," concludes Yamasoshi.
